Project Summary Esophageal sensory (afferent) nerves are essential for regulation of esophageal sensory-motor function and contribute to many clinical symptoms (dysphagia, odynophagia, nausea, non-cardiac chest pain, refractory heartburn, etc.) when stimulated or dysregulated in diseases. Therefore, the neuromodulation of esophageal afferent nerves is expected to provide relief in many conditions. Progress in advancing neuromodulatory approaches targeting esophageal afferent nerves is hampered by our limited understanding of key aspects of their neurobiology. In this three-year proposal we will focus on two such highly relevant understudied areas. First, the location of the nerve terminals within different esophageal compartments. This is essential for understanding of their role in esophageal diseases, and for design and placement of neuromodulation interfaces. Second, we will obtain data that will advance our understanding of the ion channels underlying action potential conduction in esophageal afferent nerves, which will inform development and optimization of electrical neuromodulation strategies. We will address these issues not only in the guinea pig and mouse but also in the esophagus and isolated esophageal nerves obtained from human donors. Aim 1 will elucidate the location of nerve terminals and axons of C-fiber subtypes in the esophagus. We hypothesize that the neural crest- and placodes-derived C-fibers innervate distinct esophageal tissue compartments (mucosa vs. muscle, respectively). We will address our hypothesis by selective visualization of placodes- vs. neural crest-derived axon and terminals with selective AAV-virus vector-GPF transfection in guinea pig and transgenic mice and evaluate key findings in human donor whole esophagi. In Aim 2, we will obtain the complete trascriptomes of esophageal afferent nerve types focusing on ion channels that mediate action potential conduction (especially NaVs and KVs). We hypothesize that the trascriptomes of the neural crest-derived (vagal jugular and spinal DRG) C-fiber neurons are similar but distinct from those of placodes-derived nodose C-fibers and mechanosensors (including NaVs and KVs). We will perform deep RNAseq analysis on neurons retrogradely labeled from the esophagus in TRPV1-tdTomato mice and validate the expression of Nav and Kv by qRT-PCR. We will also analyze vagal nodose and jugular neurons in Cynomolgus monkey. Aim 3 will evaluate the role of ion channels underlying action potential conduction in esophageal afferent nerve types by electrophysiology. We will initially evaluate the role of the various NaV (NaV1.1 ?NaV 1.9) subtypes, and later on KV channels. We hypothesize that conduction in esophageal afferent nerve types is mediated not only by Nav1.7, but also other tetrodotoxin (TTX)-sensitive channels, possibly distinct between the neural crest- and placodes-derived C-fibers (NaV1.1/1.6 vs. NaV1.2/1.3). This will be investigates in guinea pig and mouse innervated esophagus preparation using pharmacological tools and shRNA knockdown, and in human esophageal nerves.